Spermatogenesis occurs in a unique environment that is created by the somatic Sertoli cells. Not only do these cells bind to the developing male germ cells and spatially organize them within the seminiferous epithelium, they also secrete a large number of paracrine factors, carrier proteins, proteases and protease inhibitors. While it is assumed that these secretory products affect germ cell development, it is remarkable that to date no Sertoli cell secretory product has been proven to be required for production of fertile male gametes. This application tests the hypothesis that the secretion by Sertoli cells of the protein, Doppel, is absolutely required for development of fertile sperm. The rationale for this hypothesis comes from four observations. First, published data from two laboratories demonstrate that male mice with a knockout of the gene encoding Doppel, Prnd, are infertile because their sperm are incapable of undergoing the acrosome reaction. Second, the testis expresses substantially higher concentrations of Prnd mRNA than any other organ. Third, most of the Doppel in the testis is localized to Sertoli cells. Fourth, recent data from our laboratory indicate that Sertoli cells express very high levels of Prnd mRNA while spermatids, spermatocytes and spermatogonia do not express detectable levels of this transcript. It should be noted, however, that Prnd mRNA is also expressed in the caput epididymis of the mouse, albeit at much lower levels than in the testis. Thus, while all data identify Sertoli cells as the most likely source of the Doppel that is required for male fertility, one cannot a priori exclude a role for Doppel produced by the epididymis. The goal of this R03 application is to specifically evaluate the importance to fertility of the secretion of Doppel by Sertoli cells. We propose to generate transgenic mice that carry a floxed Prnd allele and to breed these mice to Amh-Cre mice, which express Cre recombinase specifically in Sertoli cells. The Sertoli cells of their progeny will have a conditional knockout of the Prnd gene. We also propose to breed mice with the floxed Prnd allele to EIIa-Cre mice, which express Cre recombinase as preimplantation embryos. Public Health Relevance: All cells of their progeny will have a conditional knockout of the Prnd gene. We will then compare the effects on fertility of the specific loss of Doppel secretion by Sertoli cells to the effects of the loss of Doppel secretion by all cells. By making this comparison, we will directly test the hypothesis that the secretory product of Sertoli cells, Doppel, is required for development of fertile sperm. Sertoli cells are the somatic cells that support and nourish developing male gametes. These cells bind to spermatogenic cells, spatially organize them within the seminiferous epithelium and secrete a large repertoire of products that are assumed to be required for male fertility. However, despite the obvious intimate relationship between Sertoli cells and spermatogenic cells, the crucial mechanisms by which Sertoli cells support spermatogenesis are mostly unknown. This application will determine if the Sertoli cell secretory protein, Doppel, is essential for male fertility. We propose to generate mice with a conditional knockout in Sertoli cells of the gene that encodes Doppel and to determine if loss of Doppel secretion by Sertoli cells renders male mice infertile. Completion of these proposed experiments hold promise for providing the first direct evidence that a Sertoli cell product is required for male fertility. Such evidence may open new avenues to the study of male fertility and, thus, to the treatment of the large population of infertile men that live in the United States.